Development of field test kits for the detection of barley yellow mosaic virus and barley mild mosaic virus

Abstract

Barley yellow mosaic virus (BaYMV) and barley mild mosaic virus (BaMMV) have been reported from all areas of the UK where winter barley is grown intensively. Yield loss in susceptible cultivars has been estimated at 30-50%. UK cultivars of barley have been identified which are resistant to the viruses and some are on the HGCA Recommended List. However, susceptible cultivars often give higher yields or have better quality and agronomic characteristics. Many crops are incorrectly diagnosed as suffering from minor or major trace element deficiencies when in fact the symptoms are due to barley mosaic virus. Considerable expense is incurred on agronomic inputs, attempting to solve problems which are actually due to virus infection. This expenditure would be avoided if rapid field diagnosis could be achieved. The aim of this project was to develop a field test kit for BaYMV and BaMMV, which would yield a reliable test result in 5-10 minutes.

Mice were immunised with purified BaYMV supplied by our scientific collaborators, IACR Rothamsted.  Several fusions were carried out, but all antibodies raised to BaYMV were IgM. IgM antibodies are not suitable for use in lateral flow device development. When fresh infected plant material became available and virus was purified at Central Science Laboratory (CSL), subsequent immunisations and fusions resulted in IgG clones being produced, IgG being the preferred isotype for subsequent development. One of the IgG clones was  selected as the antibody of choice, Y52, and was purified and tested by ELISA. Despite these monoclonals performing well in traditional ELISA, a suitable LFD could not be developed. The LFD's produced, when validated on the material supplied, were not sensitive enough when compared with ELISA or Taqman confirmation. No background was produced, i.e. no non-specific binding, which had been a problem with all IACR antibodies tested previously. The conclusion is that the BaYMV LFD could only be commercialised if further validation indicated that the tests are sensitive enough for field detection, on fresh material. During the last year no BaYMV samples had been received to CSL to further validate the test.

All the anti-BaMMV monoclonal antibodies supplied to us as tissue culture supernatant from IACR were IgM's, and all had an extremely low titre. These were unsuitable for either an ELISA test or LFD. Towards the end of the project, 2 further clones were obtained from a contact in France. These were purified and one is now used in an LFD.  The LFD produced detects BaMMV specifically in leaf sample within 7-10 minutes. The sensitivity of the assay was at least 10 times less sensitive than a comparative ELISA, but adequate for detection in field samples.These LFD's were sent out to various Arable Research Centre sites for field validation. One batch of devices was supplied to ARC and another batch to IACR. To date only the ACR results have been returned. The test results from the LFD's correlated well with the ELISA test on the same material.

Despite numerous events, including Cereals 2002, promoting the BaMMV LFD as well as making the test available to purchase as a Pocket Diagnnostic product, (see www.pocketdiagnostic.com), at a discounted rate for validation purposes, as well as making the test available to all the UK PHSI, no further validation on field samples has been achieved. It is believed that the BaMMV test performs adequately in the field on fresh material, but further validation is still required to build customer confidence. Unfortunately, during the last year the incidence of BaMMV and BaYMV has been low, and hence validation has not been possible. Last year only single samples were received and in that situation we advise that more sensitive methods for detection and confirmation purposes should be employed, namely ELISA and Taqman. Only when numerous samples are required at field locations would the LFD be a benefit to the user.